How a eukaryotic cell translates the small concentration difference of a chemoattractant on its surface into highly polarized intracellular responses is a fundamental question in chemotaxis. Chemoattractants are detected by G-protein coupled receptors (GPCRs). Receptor activation upon ligand binding induces the dissociation of heterotrimeric G-protein into Galpha; and Gbetagamma subunits. These subunits, in turn, modulate the function of other intracellular signaling components to generate biochemical responses that exhibit a high degree of spatial polarization if there are asymmetries in the activation of the surface receptors. The ability to translate small asymmetry in stimulation into substantial intracellular signaling polarity provides chemotaxing cells with an acute sense of direction. They are capable of orientation in very shallow gradients with as little as a 2% difference in chemoattractant concentration between the front and back of the migrating cell. Activation of G-protein-coupled chemoattractant receptors triggers dissociation of Galpha and Gbetagamma subunits. These subunits induce intracellular responses that can be highly polarized when a cell experiences a gradient of chemoattractant. Exactly how a cell achieves this amplified signal polarization is still not well understood. Here, we quantitatively measure temporal and spatial changes of receptor occupancy, G-protein activation by FRET imaging, and PIP3 levels by monitoring the dynamics of PHCrac-GFP translocation in single living cells in response to different chemoattractant fields. Our results provided the first direct evidence that G-proteins are activated to different extents on the cell surface in response to asymmetrical stimulations. A stronger, uniformly applied stimulation triggers not only a stronger G-protein activation but also a faster adaptation of downstream responses. When naive cells (which have not experienced chemoattractant) were abruptly exposed to stable cAMP gradients, G-proteins were persistently activated throughout the entire cell surface, whereas the response of PHCrac-GFP translocation surprisingly consisted of two phases, an initial transient and asymmetrical translocation around the cell membrane, followed by a second phase producing a highly polarized distribution of PHCrac-GFP. We propose a revised model of gradient sensing, suggesting an important role for locally controlled components that inhibit PI3Kinase activity (Xu et al., 2005). Chemotactic cytokines (chemokines) bind to cell surface receptors that are linked to heterotrimeric G-proteins. Upon binding to ligands, chemokine GPCR receptors activate G-proteins and modulate intracellular signaling events. Chemokines and their receptors play critical roles in many physiological processes including inflammation, allergy, tumor progression and HIV infection. Several chemokine receptors have been found in lipid rafts of membranes. Regulation of the localization of chemokine receptors may modulate the function of some, if not all, chemokine receptors (Manes et al., 2001). The goal of this project is to determine if chemokine receptor function depends on the integrity of lipid rafts and if the membrane distribution of the receptors is altered upon binding to ligands. Ligand binding to a chemokine receptor triggers signaling events through heterotrimeric G-proteins. The mechanisms underlying receptor-mediated G-protein activation in the heterogeneous microenvironments of the plasma membrane are unclear. Here, using live cell FRET imaging to detect the proximity between CXCR1-CFP and fluorescence probes that label lipid raft or non-raft microdomains, and FRAP analysis to measure the lateral diffusion of CXCR1-CFP, we found that IL-8 induces association between the receptors and lipid-raft microenvironments. Disruption of lipid rafts impaired G-protein-dependent signaling such as Ca2+ responses and PI3K activation, but had no effect on ligand-binding function and did not completely abolish ligand-induced receptor phosphorylation. Our results suggest a novel mechanism by which ligand binding to CXCR1 promotes lipid raft partitioning of receptors and facilitates activation of heterotrimeric G-proteins (Jiao et al., 2005).